This project consists of five parts, all of which center on the structure and functioning of the Na,K-ATPase. (1) Transient kinetics studies have established rate constants for the conformational transition, El-P==Greater than El-P, and have also demonstrated an ADP-stimulated hydrolysis of El-P. (2) Conditions which promote the formation of phosphoenzyme from ortho-phosphate are under study. Results support the hypothesis that the energy state of the enzyme acylphosphate is determined by factor which control access of water to the catalytic side. (3) Quantitative solubilization and partial purification of the Na,K-ATPase activity from rodent brain have been achieved utilizing a new detergent and affinity columns. The objective of this work is to examine the question of the occurrence of isozymes of the ATPase in brain. (4) The relation between structure and function in the Na,K-ATPase is being studied through the application of a battery of monoclonal antibodies which are being screened for structural specificity and functional interactions. Monoclonal antibobies are also being applied to the investigation of ATPase isozymes. (5) The Electrophorus electric organ is being investigated as a source of mRNA for the Na,K-ATPase. Preliminary studies have shown that proteins in the correct molecular weight range are coded for by the partially purified RNA preparation. The identity of the proteins will be tested immunologically. Depending on the results, further work may include the preparation of cDNA for sequencing and hybridization studies.